Publications by authors named "Stefan H Heinemann"

134 Publications

Fe-Mediated Activation of BK Channels by Rapid Photolysis of CORM-S1 Releasing CO and Fe.

ACS Chem Biol 2020 08 29;15(8):2098-2106. Epub 2020 Jul 29.

Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Str. 2, D-07745 Jena, Germany.

Heme catabolism by heme oxygenase (HO) with a decrease in intracellular heme concentration and a concomitant local release of CO and Fe has the potential to regulate BK channels. Here, we show that the iron-based photolabile CO-releasing molecule CORM-S1 [dicarbonyl-bis(cysteamine)iron(II)] coreleases CO and Fe, making it a suitable light-triggered source of these downstream products of HO activity. To investigate the impact of CO, iron, and cysteamine on BK channel activation, human Slo1 (hSlo1) was expressed in HEK293T cells and studied with electrophysiological methods. Whereas hSlo1 channels are activated by CO and even more strongly by Fe, Fe and cysteamine possess only marginal activating potency. Investigation of hSlo1 mutants revealed that Fe modulates the channels mainly through the Mg-dependent activation mechanism. Flash photolysis of CORM-S1 suits for rapid and precise delivery of Fe and CO in biological settings.
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http://dx.doi.org/10.1021/acschembio.0c00282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442670PMC
August 2020

Impact of intracellular hemin on N-type inactivation of voltage-gated K channels.

Pflugers Arch 2020 05 10;472(5):551-560. Epub 2020 May 10.

Department of Biophysics, Center for Molecular Biomedicine, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Str. 2, D-07745, Jena, Germany.

N-type inactivation of voltage-gated K channels is conferred by the N-terminal "ball" domains of select pore-forming α subunits or of auxiliary β subunits, and influences electrical cellular excitability. Here, we show that hemin impairs inactivation of K channels formed by Kv3.4 α subunits as well as that induced by the subunits Kvβ1.1, Kvβ1.2, and Kvβ3.1 when coexpressed with α subunits of the Kv1 subfamily. In Kvβ1.1, hemin interacts with cysteine and histidine residues in the N terminus (C7 and H10) with high affinity (EC 100 nM). Similarly, rapid inactivation of Kv4.2 channels induced by the dipeptidyl peptidase-like protein DPP6a is also sensitive to hemin, and the DPP6a mutation C13S eliminates this dependence. The results suggest a common mechanism for a dynamic regulation of Kv channel inactivation by heme/hemin in N-terminal ball domains of Kv α and auxiliary β subunits. Free intracellular heme therefore has the potential to regulate cellular excitability via modulation of Kv channel inactivation.
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http://dx.doi.org/10.1007/s00424-020-02386-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239824PMC
May 2020

Structural insights into heme binding to IL-36α proinflammatory cytokine.

Sci Rep 2019 11 15;9(1):16893. Epub 2019 Nov 15.

Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, D-53121, Bonn, Germany.

Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.
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http://dx.doi.org/10.1038/s41598-019-53231-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858345PMC
November 2019

Labile heme impairs hepatic microcirculation and promotes hepatic injury.

Arch Biochem Biophys 2019 09 11;672:108075. Epub 2019 Aug 11.

Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Germany; Center for Sepsis Control and Care, Jena University Hospital, Germany; Institute for Infectious Disease and Infection Control, Jena University Hospital, Germany. Electronic address:

Sepsis is a life-threatening clinical syndrome defined as a deregulated host response to infection associated with organ dysfunction. Mechanisms underlying the pathophysiology of septic liver dysfunction are incompletely understood. Among others, the iron containing tetrapyrrole heme inflicts hepatic damage when released into the circulation during systemic inflammation and sepsis. Accordingly, hemolysis and decreased concentrations of heme-scavenging proteins coincide with an unfavorable outcome of critically ill patients. As the liver is a key organ in heme metabolism and host response to infection, we investigated the impact of labile heme on sinusoidal microcirculation and hepatocellular integrity. We here provide experimental evidence that heme increases portal pressure via a mechanism that involves hepatic stellate cell-mediated sinusoidal constriction, a hallmark of microcirculatory failure under stress conditions. Moreover, heme exerts direct cytotoxicity in vitro and aggravates tissue damage in a model of polymicrobial sepsis. Heme binding by albumin, a low-affinity but high-capacity heme scavenger, attenuates heme-mediated vasoconstriction in vivo and prevents heme-mediated cytotoxicity in vitro. We demonstrate that fractions of serum albumin-bound labile heme are increased in septic patients. We propose that heme scavenging might be used therapeutically to maintain hepatic microcirculation and organ function in sepsis.
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http://dx.doi.org/10.1016/j.abb.2019.108075DOI Listing
September 2019

Membrane potential manipulation with visible flash lamp illumination of targeted microbeads.

Biochem Biophys Res Commun 2019 09 25;517(2):297-302. Epub 2019 Jul 25.

Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena & Jena University Hospital, Jena, Germany. Electronic address:

The electrical membrane potential (V) is a key dynamical variable of excitable membranes. Despite the tremendous success of optogenetic methods to modulate V with light, there are some shortcomings, such as the need of genetic manipulation and limited time resolution. Direct optical stimulation of gold nanoparticles targeted to cells is an attractive alternative because the absorbed energy heats the membrane and, thus, generates capacitive current sufficient to trigger action potentials [1, Carvalho-de-Souza et al., 2015]. However, focused laser light is required and precise location and binding of the nanoparticles cannot be assessed with a conventional microscope. We therefore examined a complementary method to manipulate V in a spatio-temporal fashion by non-focused visible flashlight stimulation (Xenon discharge lamp, 385-485 nm, ∼500 μs) of superparamagnetic microbeads. Flashlight stimulation of single beads targeted to cells resulted in transient inward currents under whole-cell patch-clamp control. The waveform of the current reflected the first time derivative of the local temperature induced by the absorbed light and subsequent heat dissipation. The maximal peak current as well as the temperature excursion scaled with the proximity to the plasma membrane. Transient illumination of light-absorbing beads, targeted to specific cellular sites via protein-protein interaction or direct micromanipulation, may provide means of rapid and spatially confined heating and electrical cell stimulation.
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http://dx.doi.org/10.1016/j.bbrc.2019.07.080DOI Listing
September 2019

Linkage Evidence for a Two-Locus Inheritance of LQT-Associated Seizures in a Multigenerational LQT Family With a Novel Loss-of-Function Mutation.

Front Neurol 2019 25;10:648. Epub 2019 Jun 25.

Department of Neurology and Experimental Neurology, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Mutations in several genes encoding ion channels can cause the long-QT (LQT) syndrome with cardiac arrhythmias, syncope and sudden death. Recently, mutations in some of these genes were also identified to cause epileptic seizures in these patients, and the sudden unexplained death in epilepsy (SUDEP) was considered to be the pathologic overlap between the two clinical conditions. For LQT-associated mutations, only few investigations reported the coincidence of cardiac dysfunction and epileptic seizures. Clinical, electrophysiological and genetic characterization of a large pedigree ( = 241 family members) with LQT syndrome caused by a 12-base-pair duplication in exon 8 of the gene duplicating four amino acids in the carboxyterminal KCNQ1 domain (dup12; p.R360_Q361dupQKQR, NM_000218.2, hg19). Electrophysiological recordings revealed no substantial KCNQ1-like currents. The mutation did not exhibit a dominant negative effect on wild-type KCNQ1 channel function. Most likely, the mutant protein was not functionally expressed and thus not incorporated into a heteromeric channel tetramer. Many LQT family members suffered from syncopes or developed sudden death, often after physical activity. Of 26 family members with LQT, seizures were present in 14 (LQTplus seizure trait). Molecular genetic analyses confirmed a causative role of the novel dup12 mutation for the LQT trait and revealed a strong link also with the LQTplus seizure trait. Genome-wide parametric multipoint linkage analyses identified a second strong genetic modifier locus for the LQTplus seizure trait in the chromosomal region 10p14. The linkage results suggest a two-locus inheritance model for the LQTplus seizure trait in which both the dup12 mutation and the 10p14 risk haplotype are necessary for the occurrence of LQT-associated seizures. The data strongly support emerging concepts that mutations may increase the risk of epilepsy, but additional genetic modifiers are necessary for the clinical manifestation of epileptic seizures.
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http://dx.doi.org/10.3389/fneur.2019.00648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6603176PMC
June 2019

Effect of Conformational Diversity on the Bioactivity of µ-Conotoxin PIIIA Disulfide Isomers.

Mar Drugs 2019 Jul 2;17(7). Epub 2019 Jul 2.

Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.

Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel Na1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at Na1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer's activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at Na1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble.
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http://dx.doi.org/10.3390/md17070390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669574PMC
July 2019

Disruption of Membrane Integrity by the Bacterium-Derived Antifungal Jagaricin.

Antimicrob Agents Chemother 2019 09 23;63(9). Epub 2019 Aug 23.

Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute, Jena, Germany

Jagaricin is a lipopeptide produced by the bacterial mushroom pathogen , the causative agent of mushroom soft rot disease. Apart from causing lesions in mushrooms, jagaricin is a potent antifungal active against human-pathogenic fungi. We show that jagaricin acts by impairing membrane integrity, resulting in a rapid flux of ions, including Ca, into susceptible target cells. Accordingly, the calcineurin pathway is required for jagaricin tolerance in the fungal pathogen Transcriptional profiling of pathogenic yeasts further revealed that jagaricin triggers cell wall strengthening, general shutdown of membrane potential-driven transport, and the upregulation of lipid transporters, linking cell envelope integrity to jagaricin action and resistance. Whereas jagaricin shows hemolytic effects, it exhibited either no or low plant toxicity at concentrations at which the growth of prevalent phytopathogenic fungi is inhibited. Therefore, jagaricin may have potential for agricultural applications. The action of jagaricin as a membrane-disrupting antifungal is promising but would require modifications for use in humans.
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http://dx.doi.org/10.1128/AAC.00707-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709453PMC
September 2019

Large-conductance Ca- and voltage-gated K channels form and break interactions with membrane lipids during each gating cycle.

Proc Natl Acad Sci U S A 2019 04 9;116(17):8591-8596. Epub 2019 Apr 9.

Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104;

Membrane depolarization and intracellular Ca promote activation of the large-conductance Ca- and voltage-gated (Slo1) big potassium (BK) channel. We examined the physical interactions that stabilize the closed and open conformations of the ion conduction gate of the human Slo1 channel using electrophysiological and computational approaches. The results show that the closed conformation is stabilized by intersubunit ion-ion interactions involving negative residues (E321 and E324) and positive residues (RKK) at the cytoplasmic ends of the transmembrane S6 segments ("RKK ring"). When the channel gate is open, the RKK ring is broken and the positive residues instead make electrostatic interactions with nearby membrane lipid oxygen atoms. E321 and E324 are stabilized by water. When the RKK residues are mutated to hydrophobic amino acids, these residues form even stronger hydrophobic interactions with the lipid tails to promote the open conformation, shifting the voltage dependence of activation to the negative direction by up to 400 mV and stabilizing the selectivity filter region. Thus, the RKK segment forms electrostatic interactions with oxygen atoms from two sources, other amino acid residues (E321/E324), and membrane lipids, depending on the gate status. Each time the channel opens and closes, the aforementioned interactions are formed and broken. This lipid-dependent Slo1 gating may explain how amphipathic signaling molecules and pharmacologically active agents influence the channel activity, and a similar mechanism may be operative in other ion channels.
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http://dx.doi.org/10.1073/pnas.1901381116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486743PMC
April 2019

ANK2 functionally interacts with KCNH2 aggravating long QT syndrome in a double mutation carrier.

Biochem Biophys Res Commun 2019 05 28;512(4):845-851. Epub 2019 Mar 28.

Department of Cardiology, Medical University Hospital Heidelberg, INF 410, D-69120, Heidelberg, Germany; DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, University of Heidelberg, INF 410, D-69120, Heidelberg, Germany. Electronic address:

Pathogenic long QT mutations often comprise high phenotypic variability and particularly variants in ANK2 (long QT syndrome 4) frequently lack QT prolongation. We sought to elucidate the genetic and functional background underlying the clinical diversity in a 3-generation family with different cardiac arrhythmias. Next-generation sequencing-based screening of patients with QT prolongation identified the index patient of the family carrying an ANK2-E1813K variant and a previously uncharacterized KCNH2-H562R mutation in a double heterozygous conformation. The patient presented with a severe clinical phenotype including a markedly prolonged QTc interval (544 ms), recurrent syncope due to Torsade de Pointes tachycardias, survived cardiopulmonary resuscitation, progressive cardiac conduction defect, and atrial fibrillation. Evaluation of other family members identified a sister and a niece solely carrying the ANK2-E1813K variant, who showed age-related conduction disease. An asymptomatic second sister solely carried the KCNH2-H562R mutation. Voltage-clamp recordings in Xenopus oocytes revealed that KCNH2-H562R subunits were non-functional but did not exert dominant-negative effects on wild-type subunits. Expression of KCNH2-H562R in HEK293 cells showed a trafficking deficiency. Co-expression of the C-terminal regulatory domain of ANK2 in Xenopus oocytes revealed that ANK2-E1813K diminished currents mediated by the combination of wild-type and H562R KCNH2 subunits. Our data suggest that ANK2 functionally interacts with KCNH2 leading to a stronger current suppression and marked aggravation of long QT syndrome in the patient carrying variants in both proteins.
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http://dx.doi.org/10.1016/j.bbrc.2019.03.162DOI Listing
May 2019

Where cone snails and spiders meet: design of small cyclic sodium-channel inhibitors.

FASEB J 2019 03 3;33(3):3693-3703. Epub 2018 Dec 3.

Toxicology and Pharmacology, Katholieke Universiteit (KU) Leuven, Campus Gasthuisberg, Leuven, Belgium.

A 13 aa residue voltage-gated sodium (Na) channel inhibitor peptide, Pn, containing 2 disulfide bridges was designed by using a chimeric approach. This approach was based on a common pharmacophore deduced from sequence and secondary structural homology of 2 Na inhibitors: Conus kinoshitai toxin IIIA, a 14 residue cone snail peptide with 3 disulfide bonds, and Phoneutria nigriventer toxin 1, a 78 residue spider toxin with 7 disulfide bonds. As with the parent peptides, this novel Na channel inhibitor was active on Na1.2. Through the generation of 3 series of peptide mutants, we investigated the role of key residues and cyclization and their influence on Na inhibition and subtype selectivity. Cyclic PnCS1, a 10 residue peptide cyclized via a disulfide bond, exhibited increased inhibitory activity toward therapeutically relevant Na channel subtypes, including Na1.7 and Na1.9, while displaying remarkable serum stability. These peptides represent the first and the smallest cyclic peptide Na modulators to date and are promising templates for the development of toxin-based therapeutic agents.-Peigneur, S., Cheneval, O., Maiti, M., Leipold, E., Heinemann, S. H., Lescrinier, E., Herdewijn, P., De Lima, M. E., Craik, D. J., Schroeder, C. I., Tytgat, J. Where cone snails and spiders meet: design of small cyclic sodium-channel inhibitors.
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http://dx.doi.org/10.1096/fj.201801909RDOI Listing
March 2019

Modulation of K channel N-type inactivation by sulfhydration through hydrogen sulfide and polysulfides.

Pflugers Arch 2019 04 10;471(4):557-571. Epub 2018 Nov 10.

Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Str. 2, 07745, Jena, Germany.

Fast N-type inactivation of voltage-gated K (Kv) channels is important in fine-tuning of cellular excitability. To serve diverse cellular needs, N-type inactivation is regulated by numerous mechanisms. Here, we address how reactive sulfur species-the gaseous messenger HS and polysulfides-affect N-type inactivation of the mammalian Kv channels Kv1.4 and Kv3.4. In both channels, the HS donor NaHS slowed down inactivation with varying potency depending on the "aging" of NaHS solution. Polysulfides were > 1000 times more effective than NaHS with the potency increasing with the number of sulfur atoms (NaS < NaS < NaS). In Kv1.4, C13 in the N-terminal ball domain mediates the slowing of inactivation. In recombinant protein exposed to NaHS or NaS, a sulfur atom is incorporated at C13 in the protein. In Kv3.4, the N terminus harbors two cysteine residues (C6, C24), and C6 is of primary importance for channel regulation by HS and polysulfides, with a minor contribution from C24. To fully eliminate the dependence of N-type inactivation on sulfhydration, both cysteine residues must be removed (C6S:C24S). Sulfhydration of a single cysteine residue in the ball-and-chain domain modulates the speed of inactivation but does not remove it entirely. In both Kv1.4 and Kv3.4, polysulfides affected the N-terminal cysteine residues when assayed in the whole-cell configuration; on-cell recordings confirmed that polysulfides also modulate K channel inactivation with undisturbed cytosol. These findings have collectively identified reactive sulfur species as potent modulators of N-type inactivation in mammalian Kv channels.
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http://dx.doi.org/10.1007/s00424-018-2233-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086210PMC
April 2019

Studies into Slo1 K channels and their ligand docosahexaenoic acid in murine sepsis to delineate off-target effects of immunonutrition.

Life Sci 2018 Jun 21;203:112-120. Epub 2018 Apr 21.

Department of Anaesthesiology and Intensive Care Medicine, Jena University Hospital, Jena, Germany; Centre for Innovation Competence Septomics, Friedrich Schiller University Jena, Jena, Germany; Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany. Electronic address:

Aims: Studies on omega-3 fatty acids, including docosahexaenoic acid (DHA), reveal diverging results: Their intake is recommended in cardiovascular disease and major surgery, while evidence argues against use in septic patients. DHA mediates its blood-pressure-lowering effect through Slo1 channels that are expressed on cardiovascular and immune cells. We hypothesised that conflicting effects of immunonutrition could be explained by the influence of omega-3 fatty acids on systemic blood pressure or immune effector cells through Slo1.

Main Methods: The effect of DHA on blood pressure was analysed in septic wild-type (WT) mice. Septic WT and Slo1 knockout (KO) mice were compared regarding survival, clinical presentation, haematology, cytokine release and bacterial burden. Cytokine expression and release of bone marrow derived macrophages (BMDM) from WT and Slo1 KO mice was assessed in response to LPS.

Key Findings: The significant blood-pressure-lowering effect of DHA in healthy animals was blunted in already hypotensive septic mice. Septic Slo1 KO mice displayed moderately lower bacterial burden in blood and lungs compared with WT, which did not translate into improved survival. Slo1 KO BMDM presented lower IL-6 levels in response to LPS, an effect that was abolished in the presence of DHA. More importantly, the strong inhibitory effect of DHA on IL-6 release was also observed in Slo1 KO BMDM.

Significance: The controversial effects of immunonutrition in sepsis are unlikely to be primarily explained by the influence of DHA on blood pressure or effects on immune response mediated through Slo1 channels.
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http://dx.doi.org/10.1016/j.lfs.2018.04.031DOI Listing
June 2018

CO-independent modification of K channels by tricarbonyldichlororuthenium(II) dimer (CORM-2).

Eur J Pharmacol 2017 Nov 5;815:33-41. Epub 2017 Oct 5.

Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena & Jena University Hospital, Hans-Knöll-Str. 2, D-07745 Jena, Germany. Electronic address:

Although toxic when inhaled in high concentrations, the gas carbon monoxide (CO) is endogenously produced in mammals, and various beneficial effects are reported. For potential medicinal applications and studying the molecular processes underlying the pharmacological action of CO, so-called CO-releasing molecules (CORMs), such as tricabonyldichlororuthenium(II) dimer (CORM-2), have been developed and widely used. Yet, it is not readily discriminated whether an observed effect of a CORM is caused by the released CO gas, the CORM itself, or any of its intermediate or final breakdown products. Focusing on Ca- and voltage-dependent K channels (K1.1) and voltage-gated K channels (Kv1.5, Kv11.1) relevant for cardiac safety pharmacology, we demonstrate that, in most cases, the functional impacts of CORM-2 on these channels are not mediated by CO. Instead, when dissolved in aqueous solutions, CORM-2 has the propensity of forming Ru(CO) adducts, preferentially to histidine residues, as demonstrated with synthetic peptides using mass-spectrometry analysis. For K1.1 channels we show that H365 and H394 in the cytosolic gating ring structure are affected by CORM-2. For Kv11.1 channels (hERG1) the extracellularly accessible histidines H578 and H587 are CORM-2 targets. The strong CO-independent action of CORM-2 on Kv11.1 and Kv1.5 channels can be completely abolished when CORM-2 is applied in the presence of an excess of free histidine or human serum albumin; cysteine and methionine are further potential targets. Off-site effects similar to those reported here for CORM-2 are found for CORM-3, another ruthenium-based CORM, but are diminished when using iron-based CORM-S1 and absent for manganese-based CORM-EDE1.
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http://dx.doi.org/10.1016/j.ejphar.2017.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662205PMC
November 2017

Impact of higher-order heme degradation products on hepatic function and hemodynamics.

J Hepatol 2017 08 12;67(2):272-281. Epub 2017 Apr 12.

Department of Anesthesiology and Intensive Care Medicine/Center for Sepsis Control and Care, Jena University Hospital, Germany. Electronic address:

Background & Aims: Biliverdin and bilirubin were previously considered end products of heme catabolism; now, however, there is evidence for further degradation to diverse bioactive products. Z-BOX A and Z-BOX B arise upon oxidation with unknown implications for hepatocellular function and integrity. We studied the impact of Z-BOX A and B on hepatic functions and explored their alterations in health and cholestatic conditions.

Methods: Functional implications and mechanisms were investigated in rats, hepatocytic HepG2 and HepaRG cells, human immortalized hepatocytes, and isolated perfused livers. Z-BOX A and B were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in acute and acute-on-chronic liver failure and hereditary unconjugated hyperbilirubinemia.

Results: Z-BOX A and B are found in similar amounts in humans and rodents under physiological conditions. Serum concentrations increased ∼20-fold during cholestatic liver failure in humans (p<0.001) and in hereditary deficiency of bilirubin glucuronidation in rats (p<0.001). Pharmacokinetic studies revealed shorter serum half-life of Z-BOX A compared to its regio-isomer Z-BOX B (p=0.035). While both compounds were taken up by hepatocytes, Z-BOX A was enriched ∼100-fold and excreted in bile. Despite their reported vasoconstrictive properties in the brain vasculature, BOXes did not affect portal hemodynamics. Both Z-BOX A and B showed dose-dependent cytotoxicity, affected the glutathione redox state, and differentially modulated activity of Rev-erbα and Rev-erbβ. Moreover, BOXes-triggered remodeling of the hepatocellular cytoskeleton.

Conclusions: Our data provide evidence that higher-order heme degradation products, namely Z-BOX A and B, impair hepatocellular integrity and might mediate intra- and extrahepatic cytotoxic effects previously attributed to hyperbilirubinemia.

Lay Summary: Degradation of the blood pigment heme yields the bile pigment bilirubin and the oxidation products Z-BOX A and Z-BOX B. Serum concentrations of these bioactive molecules increase in jaundice and can impair liver function and integrity. Amounts of Z-BOX A and Z-BOX B that are observed during liver failure in humans have profound effects on hepatic function when added to cultured liver cells or infused into healthy rats.
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http://dx.doi.org/10.1016/j.jhep.2017.03.037DOI Listing
August 2017

Non-photonic sensing of membrane-delimited reactive species with a Na channel protein containing selenocysteine.

Sci Rep 2017 04 5;7:46003. Epub 2017 Apr 5.

Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena &Jena University Hospital, Jena, Germany.

Photonic experiments are of key importance in life sciences but light-induced side effects are serious confounding factors. Here we introduce roNa2, an engineered voltage-gated Na channel harboring a selenocysteine in its inactivation motif, as a non-photonic, sensitive, gateable, and reversible sensor for membrane-delimited reactive species. roNa2 allows for the assessment of chemical modification induced in fluorescence microscopy settings with high sensitivity and time resolution and it demonstrates the usefulness of ion channels as highly sensitive reporters of membrane processes.
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http://dx.doi.org/10.1038/srep46003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381000PMC
April 2017

Synthesis and solution stability of water-soluble κN,κO-bis(3,5-dimethylpyrazolyl)ethanol manganese(i) tricarbonyl bromide (CORM-ONN1).

Dalton Trans 2017 Jan;46(5):1684-1693

Institute of Inorganic and Analytical Chemistry, Friedrich Schiller University (FSU), Humboldstraße 8, D-07743 Jena, Germany.

The reaction of (OC)MnBr with bis(3,5-dimethyl-1-pyrazolyl)methane yields [{(Pz)CH}Mn(CO)Br] (1). The use of tridentate heteroscorpionates such as bis(3,5-dimethyl-1-pyrazolyl)acetic acid and 2,2-bis(3,5-dimethyl-1-pyrazolyl)ethanol leads to the formation of mononuclear [(OC)Mn{(Pz)CH-CO}] (2) and [(OC)Mn{(Pz)CH-CHOH}]Br (3, CORM-ONN1). Salt-like photoCORM 3 is soluble in aqueous media up to a concentration of 200 μM, non-toxic up to an approx. 65 μM solution and releases all carbonyl ligands upon irradiation. The molecular complexes 1 and 2 are insoluble in aqueous solutions. CORM-ONN1 (3) slowly degrades in methanol yielding iCORM 4, consisting of the complex cation [{(Pz)CH-CHOH}{(Pz)CH-CHO}Mn] and the [Mn(CO)] counter anion with the cations linked to a dimeric unit by intermolecular hydrogen bridges between the alcohol and alkoxide functionalities.
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http://dx.doi.org/10.1039/c6dt03551eDOI Listing
January 2017

Subtype-specific block of voltage-gated K channels by μ-conopeptides.

Biochem Biophys Res Commun 2017 Jan 2;482(4):1135-1140. Epub 2016 Dec 2.

Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena & Jena University Hospital, Hans-Knöll-Straße 2, D-07745 Jena, Germany. Electronic address:

The neurotoxic cone snail peptide μ-GIIIA specifically blocks skeletal muscle voltage-gated sodium (Na1.4) channels. The related conopeptides μ-PIIIA and μ-SIIIA, however, exhibit a wider activity spectrum by also inhibiting the neuronal Na channels Na1.2 and Na1.7. Here we demonstrate that those μ-conopeptides with a broader target range also antagonize select subtypes of voltage-gated potassium channels of the K1 family: μ-PIIIA and μ-SIIIA inhibited K1.1 and K1.6 channels in the nanomolar range, while being inactive on subtypes K1.2-1.5 and K2.1. Construction and electrophysiological evaluation of chimeras between K1.5 and K1.6 revealed that these toxins block K channels involving their pore regions; the subtype specificity is determined in part by the sequence close to the selectivity filter but predominantly by the so-called turret domain, i.e. the extracellular loop connecting the pore with transmembrane segment S5. Conopeptides μ-SIIIA and μ-PIIIA, thus, are not specific for Na channels, and the known structure of some K channel subtypes may provide access to structural insight into the molecular interaction between μ-conopeptides and their target channels.
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http://dx.doi.org/10.1016/j.bbrc.2016.11.170DOI Listing
January 2017

Bioactivity of topologically confined gramicidin A dimers.

Bioorg Med Chem 2017 01 9;25(1):261-268. Epub 2016 Nov 9.

Friedrich Schiller University Jena, Institute of Organic Chemistry and Macromolecular Chemistry, Humboldtstr. 10, D-07743 Jena, Germany. Electronic address:

The d-/l-peptide gramicidin A (gA) is well known as a pivotal ion channel model and shows a broad spectrum of bioactivities such as antibiosis, antimalarial activity, as well as hemolysis. We applied inter-chain disulfide bonds to constrain the conformational freedom of gA into parallel and antiparallel dimeric topologies. Albeit the constructs were not found to be monoconformational, CD- and IR-spectroscopic studies suggested that this strategy indeed restricted the conformational space of the d-/l-peptide construct, and that β-helical secondary structures prevail. Correlative testing of gA dimers in antimicrobial, antimalarial, and ion conduction assays suggested that the tail-to-tail antiparallel single stranded β helix dominantly mediates the bioactivity of gA. Other conformers are unlikely to contribute to these activities. From these investigations, only weakly ion conducting gA dimers were identified that retained nM antimalarial activity.
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http://dx.doi.org/10.1016/j.bmc.2016.10.033DOI Listing
January 2017

Atomic determinants of BK channel activation by polyunsaturated fatty acids.

Proc Natl Acad Sci U S A 2016 11 14;113(48):13905-13910. Epub 2016 Nov 14.

Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104;

Docosahexaenoic acid (DHA), a polyunsaturated ω-3 fatty acid enriched in oily fish, contributes to better health by affecting multiple targets. Large-conductance Ca- and voltage-gated Slo1 BK channels are directly activated by nanomolar levels of DHA. We investigated DHA-channel interaction by manipulating both the fatty acid structure and the channel composition through the site-directed incorporation of unnatural amino acids. Electrophysiological measurements show that the para-group of a Tyr residue near the ion conduction pathway has a critical role. To robustly activate the channel, ionization must occur readily by a fatty acid for a good efficacy, and a long nonpolar acyl tail with a Z double bond present at the halfway position for a high affinity. The results suggest that DHA and the channel form an ion-dipole bond to promote opening and demonstrate the channel druggability. DHA, a marine-derived nutraceutical, represents a promising lead compound for rational drug design and discovery.
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http://dx.doi.org/10.1073/pnas.1615562113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137720PMC
November 2016

Molecular Insights into the Mechanism of Calmodulin Inhibition of the EAG1 Potassium Channel.

Structure 2016 Oct 8;24(10):1742-1754. Epub 2016 Sep 8.

Instituto de Biologia Molecular e Celular, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal. Electronic address:

The human EAG1 potassium channel belongs to the superfamily of KCNH voltage-gated potassium channels that have roles in cardiac repolarization and neuronal excitability. EAG1 is strongly inhibited by Ca/calmodulin (CaM) through a mechanism that is not understood. We determined the binding properties of CaM with each one of three previously identified binding sites (BDN, BDC1, and BDC2), analyzed binding to protein stretches that include more than one site, and determined the effect of neighboring globular domains on the binding properties. The determination of the crystal structure of CaM bound to BDC2 shows the channel fragment interacting with only the C lobe of calmodulin and adopting an unusual bent conformation. Based on this structure and on a functional and biochemical analysis of mutants, we propose a model for the mechanism of inhibition whereby the local conformational change induced by CaM binding at BDC2 lies at the basis of channel modulation.
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http://dx.doi.org/10.1016/j.str.2016.07.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5176025PMC
October 2016

Molecular interaction of δ-conopeptide EVIA with voltage-gated Na(+) channels.

Biochim Biophys Acta 2016 09 15;1860(9):2053-63. Epub 2016 Jun 15.

Clemens-Schöpf-Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Str. 4, D-64287 Darmstadt, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.bbagen.2016.06.013DOI Listing
September 2016

Stereospecific capillary electrophoresis assays using pentapeptide substrates for the study of Aspergillus nidulans methionine sulfoxide reductase A and mutant enzymes.

Electrophoresis 2016 07 8;37(14):2083-90. Epub 2016 Jun 8.

Department of Pharmaceutical Chemistry, Friedrich Schiller University Jena, Jena, Germany.

Stereospecific capillary electrophoresis-based methods for the analysis of methionine sulfoxide [Met(O)]-containing pentapeptides were developed in order to investigate the reduction of Met(O)-containing peptide substrates by recombinant Aspergillus nidulans methionine sulfoxide reductase A (MsrA) as well as enzymes carrying mutations in position Glu99 and Asp134. The separation of the diastereomers of the N-acetylated, C-terminally 2,4-dinitrophenyl (Dnp)-labeled pentapeptides ac-Lys-Phe-Met(O)-Lys-Lys-Dnp, ac-Lys-Asp-Met(O)-Asn-Lys-Dnp and ac-Lys-Asn-Met(O)-Asp-Lys-Dnp was achieved in 50 mM Tris-HCl buffers containing sulfated β-CD in fused-silica capillaries, while the diastereomer separation of ac-Lys-Asp-Met(O)-Asp-Lys-Dnp was achieved by sulfated β-CD-mediated MEKC. The methods were validated with regard to range, linearity, accuracy, limits of detection and quantitation as well as precision. Subsequently, the substrates were incubated with wild-type MsrA and three mutants in the presence of dithiothreitol as reductant. Wild-type MsrA displayed the highest activity towards all substrates compared to the mutants. Substitution of Glu99 by Gln resulted in the mutant with the lowest activity towards all substrates except for ac-Lys-Asn-Met(O)-Asp-Lys-Dnp, while replacement Asn for Asp134 lead to a higher activity towards ac-Lys-Asp-Met(O)-Asn-Lys-Dnp compared with the Glu99 mutant. The mutant with Glu instead of Asp134 was the most active among the mutant enzymes. Molecular modeling indicated that the conserved Glu99 residue is buried in the Met-S-(O) groove, which might contribute to the correct placing of substrates and, consequently, to the catalytic activity of MsrA, while Asp134 did not form hydrogen bonds with the substrates but only within the enzyme.
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http://dx.doi.org/10.1002/elps.201600181DOI Listing
July 2016

Human EAG channels are directly modulated by PIP2 as revealed by electrophysiological and optical interference investigations.

Sci Rep 2016 Mar 23;6:23417. Epub 2016 Mar 23.

Key Laboratory of Systems Biomedicine (Ministry of Education), Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China.

Voltage-gated ether à go-go (EAG) K(+) channels are expressed in various types of cancer cells and also in the central nervous system. Aberrant overactivation of human EAG1 (hEAG1) channels is associated with cancer and neuronal disorders such as Zimmermann-Laband and Temple-Baraitser syndromes. Although hEAG1 channels are recognized as potential therapeutic targets, regulation of their functional properties is only poorly understood. Here, we show that the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is a potent inhibitory gating modifier of hEAG1 channels. PIP2 inhibits the channel activity by directly binding to a short N-terminal segment of the channel important for Ca(2+)/calmodulin (CaM) binding as evidenced by bio-layer interferometry measurements. Conversely, depletion of endogenous PIP2 either by serotonin-induced phospholipase C (PLC) activation or by a rapamycin-induced translocation system enhances the channel activity at physiological membrane potentials, suggesting that PIP2 exerts a tonic inhibitory influence. Our study, combining electrophysiological and direct binding assays, demonstrates that hEAG1 channels are subject to potent inhibitory modulation by multiple phospholipids and suggests that manipulations of the PIP2 signaling pathway may represent a strategy to treat hEAG1 channel-associated diseases.
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http://dx.doi.org/10.1038/srep23417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804213PMC
March 2016

CORM-EDE1: A Highly Water-Soluble and Nontoxic Manganese-Based photoCORM with a Biogenic Ligand Sphere.

Inorg Chem 2016 Jan 16;55(1):104-13. Epub 2015 Dec 16.

Institute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena , Humboldtstraße, 8, D-07743 Jena, Germany.

[Mn(CO)5Br] reacts with cysteamine and 4-amino-thiophenyl with a ratio of 2:3 in refluxing tetrahydrofuran to the complexes of the type [{(OC)3Mn}2(μ-SCH2CH2NH3)3]Br2 (1, CORM-EDE1) and [{(OC)3Mn}2(μ-SC6H4-4-NH3)3]Br2 (2, CORM-EDE2). Compound 2 precipitates during refluxing of the tetrahydrofuran solution as a yellow solid whereas 1 forms a red oil that slowly solidifies. Recrystallization of 2 from water yields the HBr-free complex [{(OC)3Mn}2(μ-S-C6H4-4-NH2)2(μ-SC6H4-4-NH3)] (3). The n-propylthiolate ligand (which is isoelectronic to the bridging thiolate of 1) leads to the formation of the di- and tetranuclear complexes [(OC)4Mn(μ-S-nPr)2]2 and [(OC)3Mn(μ-S-nPr)]4. CORM-EDE1 possesses ideal properties to administer carbon monoxide to biological and medicinal tissues upon irradiation (photoCORM). Isolated crystalline CORM-EDE1 can be handled at ambient and aerobic conditions. This complex is nontoxic, highly soluble in water, and indefinitely stable therein in the absence of air and phosphate buffer. CORM-EDE1 is stable as frozen stock in aqueous solution without any limitations, and these stock solutions maintain their CO release properties. The reducing dithionite does not interact with CORM-EDE1, and therefore, the myoglobin assay represents a valuable tool to study the release kinetics of this photoCORM. After CO liberation, the formation of MnHPO4 in aqueous buffer solution can be verified.
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http://dx.doi.org/10.1021/acs.inorgchem.5b01904DOI Listing
January 2016

Cold-aggravated pain in humans caused by a hyperactive NaV1.9 channel mutant.

Nat Commun 2015 Dec 8;6:10049. Epub 2015 Dec 8.

Institute of Human Genetics, Jena University Hospital, 07743 Jena, Germany.

Gain-of-function mutations in the human SCN11A-encoded voltage-gated Na(+) channel NaV1.9 cause severe pain disorders ranging from neuropathic pain to congenital pain insensitivity. However, the entire spectrum of the NaV1.9 diseases has yet to be defined. Applying whole-exome sequencing we here identify a missense change (p.V1184A) in NaV1.9, which leads to cold-aggravated peripheral pain in humans. Electrophysiological analysis reveals that p.V1184A shifts the voltage dependence of channel opening to hyperpolarized potentials thereby conferring gain-of-function characteristics to NaV1.9. Mutated channels diminish the resting membrane potential of mouse primary sensory neurons and cause cold-resistant hyperexcitability of nociceptors, suggesting a mechanistic basis for the temperature dependence of the pain phenotype. On the basis of direct comparison of the mutations linked to either cold-aggravated pain or pain insensitivity, we propose a model in which the physiological consequence of a mutation, that is, augmented versus absent pain, is critically dependent on the type of NaV1.9 hyperactivity.
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http://dx.doi.org/10.1038/ncomms10049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686659PMC
December 2015

Ca(2+)/calmodulin regulates Kvβ1.1-mediated inactivation of voltage-gated K(+) channels.

Sci Rep 2015 Oct 21;5:15509. Epub 2015 Oct 21.

Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena &Jena University Hospital, Hans-Knöll-Str. 2, D-07745 Jena, Germany.

A-type K(+) channels open on membrane depolarization and undergo subsequent rapid inactivation such that they are ideally suited for fine-tuning the electrical signaling in neurons and muscle cells. Channel inactivation mostly follows the so-called ball-and-chain mechanism, in which the N-terminal structures of either the K(+) channel's α or β subunits occlude the channel pore entry facing the cytosol. Inactivation of Kv1.1 and Kv1.4 channels induced by Kvβ1.1 subunits is profoundly decelerated in response to a rise in the intracellular Ca(2+) concentration, thus making the affected channel complexes negative feedback regulators to limit neuronal overexcitation. With electrophysiological and biochemical experiments we show that the Ca(2+) dependence is gained by binding of calmodulin to the "chain" segment of Kvβ1.1 thereby compromising the mobility of the inactivation particle. Furthermore, inactivation regulation via Ca(2+)/calmodulin does not interfere with the β subunit's enzymatic activity as an NADPH-dependent oxidoreductase, thus rendering the Kvβ1.1 subunit a multifunctional receptor that integrates cytosolic signals to be transduced to altered electrical cellular activity.
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http://dx.doi.org/10.1038/srep15509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614385PMC
October 2015

Reactive species modify NaV1.8 channels and affect action potentials in murine dorsal root ganglion neurons.

Pflugers Arch 2016 Jan 17;468(1):99-110. Epub 2015 Sep 17.

Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena & Jena University Hospital, Hans-Knöll-Str. 2, 07745, Jena, Germany.

Dorsal root ganglion (DRG) neurons are important relay stations between the periphery and the central nervous system and are essential for somatosensory signaling. Reactive species are produced in a variety of physiological and pathophysiological conditions and are known to alter electric signaling. Here we studied the influence of reactive species on the electrical properties of DRG neurons from mice with the whole-cell patch-clamp method. Even mild stress induced by either low concentrations of chloramine-T (10 μM) or low-intensity blue light irradiation profoundly diminished action potential frequency but prolonged single action potentials in wild-type neurons. The impact on evoked action potentials was much smaller in neurons deficient of the tetrodotoxin (TTX)-resistant voltage-gated sodium channel NaV1.8 (NaV1.8(-/-)), the channel most important for the action potential upstroke in DRG neurons. Low concentrations of chloramine-T caused a significant reduction of NaV1.8 peak current and, at higher concentrations, progressively slowed down inactivation. Blue light had a smaller effect on amplitude but slowed down NaV1.8 channel inactivation. The observed effects were less apparent for TTX-sensitive NaV channels. NaV1.8 is an important reactive-species-sensitive component in the electrical signaling of DRG neurons, potentially giving rise to loss-of-function and gain-of-function phenomena depending on the type of reactive species and their effective concentration and time of exposure.
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http://dx.doi.org/10.1007/s00424-015-1735-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165275PMC
January 2016

Structure Elucidation and Activity of Kolossin A, the D-/L-Pentadecapeptide Product of a Giant Nonribosomal Peptide Synthetase.

Angew Chem Int Ed Engl 2015 Aug 26;54(35):10352-5. Epub 2015 Jun 26.

Friedrich-Schiller-Universität, Institut für Organische Chemie und Makromolekulare Chemie, Humboldtstr. 10, 07743 Jena (Germany).

The largest continuous bacterial nonribosomal peptide synthetase discovered so far is described. It consists of 15 consecutive modules arising from an uninterrupted, fully functional gene in the entomopathogenic bacterium Photorhabdus luminescens. The identification of its cryptic biosynthesis product was achieved by using a combination of genome analysis, promoter exchange, isotopic labeling experiments, and total synthesis of a focused collection of peptide candidates. Although it belongs to the growing class of D-/ L-peptide natural products, the encoded metabolite kolossin A was found to be largely devoid of antibiotic activity and is likely involved in interspecies communication. A stereoisomer of this peculiar natural product displayed high activity against Trypanosoma brucei rhodesiense, a recalcitrant parasite that causes the deadly disease African sleeping sickness.
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http://dx.doi.org/10.1002/anie.201502835DOI Listing
August 2015

Heterologous expression of NaV1.9 chimeras in various cell systems.

Pflugers Arch 2015 Dec 29;467(12):2423-35. Epub 2015 Apr 29.

Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Straße 2, D-07745, Jena, Germany.

SCN11A encodes the voltage-gated sodium channel NaV1.9, which deviates most strongly from the other eight NaV channels expressed in mammals. It is characterized by resistance to the prototypic NaV channel blocker tetrodotoxin and exhibits slow activation and inactivation gating. Its expression in dorsal root ganglia neurons suggests a role in motor or pain signaling functions as also recently demonstrated by the occurrence of various mutations in human SCN11A leading to altered pain sensation syndromes. The systematic investigation of human NaV1.9, however, is severely hampered because of very poor heterologous expression in host cells. Using patch-clamp and two-electrode voltage-clamp methods, we show that this limitation is caused by the C-terminal structure of NaV1.9. A chimera of NaV1.9 harboring the C terminus of NaV1.4 yields functional expression not only in neuronal cells but also in non-excitable cells, such as HEK 293T or Xenopus oocytes. The major functional difference of the chimeric channel with respect to NaV1.9 is an accelerated activation and inactivation. Since the entire transmembrane domain is preserved, it is suited for studying pharmacological properties of the channel and the functional impact of disease-causing mutations. Moreover, we demonstrate how mutation S360Y makes NaV1.9 channels sensitive to tetrodotoxin and saxitoxin and that the unusual slow open-state inactivation of NaV1.9 is also mediated by the IFM (isoleucine-phenylalanine-methionine) inactivation motif located in the linker connecting domains III and IV.
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http://dx.doi.org/10.1007/s00424-015-1709-1DOI Listing
December 2015